The salient biochemical and genetic factors involved in the regulation of in vitro enterotoxin biosynthesis by V. cholerae are under investigation. Optimal conditions for enterotoxin production in a chemically defined medium on a mult-liter scale have been developed. Enterotoxin from such crude culture filtrates is concentrated and purified in yields above 50 percent by relatively simple procedures. This enterotoxin has biological (Lb, BD, and Lipocyte) specific activity equal to or better than that obtained by more complicated isolation procedures. The entire production and purification technique has been miniaturized to accommodate the synthesis and isolation of pure radioactively labeled toxin. The labeled toxin will be employed in conjunction with immunosorbent chromatography as a radioimmunoassay for cholera enterotoxin. The feasibility of using such a system to detect cross reacting enerotoxin(s) of pathogenic E. coli will be evaluated. Development of a defined culture medium which may facilitate successful isolation and purification of E. coli enterotoxin(s) is also underway.